As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. ![]() We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Previous studies showed that these isomers might originate from a proline cis/ trans isomerization in one Fab subunit of the tsAb. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). ![]() ![]() However, these highly complex mAb formats pose significant analytical challenges. Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs).
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